Preexisting autoantibodies predict efficacy of oral insulin to cure autoimmune diabetes in combination with anti-CD3

Abstract

We have previously developed a combination therapy (CT) using anti-CD3 monoclonal antibodies together with islet-(auto)antigen immunizations that can more efficiently reverse type 1 diabetes (T1D) than either entity alone. However, clinical translation of antigen-specific therapies in general is hampered by the lack of biomarkers that could be used to optimize the modalities of antigen delivery and to predict responders from nonresponders. To support the rapid identification of candidate biomarkers, we systematically evaluated multiple variables in a mathematical disease model. The in silico predictions were validated by subsequent laboratory data in NOD mice with T1D that received anti-CD3/oral insulin CT. Our study shows that higher anti-insulin autoantibody levels at diagnosis can distinguish responders and nonresponders among recipients of CT exquisitely well. In addition, early posttreatment changes in proinflammatory cytokines were indicative of long-term remission. Coadministration of oral insulin improved and prolonged the therapeutic efficacy of anti-CD3 therapy, and long-term protection was achieved by maintaining elevated insulin-specific regulatory T cell numbers that efficiently lowered diabetogenic effector memory T cells. Our validation of preexisting autoantibodies as biomarkers to distinguish future responders from nonresponders among recipients of oral insulin provides a compelling and mechanistic rationale to more rapidly translate anti-CD3/oral insulin CT for human T1D.

FIG. 1.

Circulating biomarkers predict long-term efficacy of anti-CD3 antibody/oral insulin CT. A: Mouse IAA levels are efficient type 0 biomarkers. After new-onset of T1D, NOD females were randomized to receive anti-CD3 treatment with or without oral insulin. Before treatment randomization, the serum of each individual NOD mouse was collected to measure IAA levels in a blinded fashion. At 15 weeks posttreatment, NOD mice with a BGV <250 mg/dL were considered protected. Data show the results obtained in individual mice (n = 8–21 per group). ns, P > 0.05. B: Serum levels of pro- and anti-inflammatory cytokines in treated NOD mice. Sera from NOD mice treated with anti-CD3 alone or anti-CD3/oral insulin (CT) were collected 2 weeks after treatment started. The concentration of each cytokine was measured by Bioplex assay. At 15 weeks posttreatment, NOD mice with a BGV <250 mg/dL were considered protected. Data show the results obtained in individual mice (n = 7–23 per group). GM-CSF, granulocyte-macrophage colony-stimulating factor.

FIG. 2.

Oral insulin immunization is required to reverse severe T1D with low-dose anti-CD3. A: BGVs of individual NOD mice treated after new-onset T1D with CD3-specific antibody alone or in combination with oral insulin. Newly diagnosed diabetic female NOD mice were randomized into 12 groups to receive 3, 7.5, 15, 30, 45, or 75 μg anti-CD3 antibody alone or in combination with oral insulin. The symbols represent the BGVs of each individual mouse for up to 16 weeks after treatment started (n = 8–36 per group). Open and closed circles discriminate protected and non-protected animals at 16 weeks posttreatment initiation. B: Overtly diabetic NOD mice were treated with anti-CD3 (5 or 10 μg) daily for 3 days alone or in conjunction with insulin feedings (0.5 mg) twice a week for 5 consecutive weeks. Remission of established T1D was followed in each group. Mice were considered protected if their BGV was <250 mg/dL at 15 weeks posttreatment. The BGV and age at onset of each individual NOD mouse treated with either 30 or 15 μg anti-CD3 antibody in combination or not with oral insulin are plotted on the graphs (n = 35–62 mice per group). Mice with the most severe T1D (characterized by an early onset and higher BGVs at diagnosis) are distinguished from mice with less severe T1D by an arbitrary dark line. The percentage of responders within the most severe or less severe T1D groups at diagnosis are calculated and displayed on each side of the arbitrary dark line.

FIG. 3.

CD3 modulation is required for synergy between anti-CD3 and oral insulin treatments. A and B: In vivo TCR-αβ modulation 5, 24, and 48 h after injection with non–FcR binding CD3-specific antibody, 145–2C11 F(ab’)₂, at 2.5 or 5 μg/day for 3 consecutive days. Data are mean ± SEM of n = 6 individual mice per group. C: The absolute number (± SEM) of CD4⁺, CD8⁺, CD4⁺Foxp3⁺, and CD19⁺ cells was measured in the PLN of NOD mice treated with non–FcR binding CD3-specific antibody, 145–2C11 F(ab’)₂, at 2.5 or 5 μg/day for 3 consecutive days. D: Mean proportion (± SEM) of CD4⁺Foxp3⁺ T cells in the PLN was analyzed by fluorescence-activated cell sorter on days 0 (nontreated), 1, 3, 6, and 8 after non–FcR binding anti-CD3 treatment (2.5 or 5 μg/day for 3 consecutive days). Data are means ± SEM of two independent experiments with at least three mice per time point. E: Mean proportion (± SEM) of CD4⁺Foxp3⁺ T cells in various organs at 2 weeks posttreatment with 30 μg anti-CD3 antibody alone (CD3) or in combination with oral insulin (CT). Data show the results obtained in individual mice. n = 6–12 in one representative experiment of three. MLN, mesenteric lymph node.

FIG. 4.

Oral insulin synergizes with anti-CD3 therapy to prolong long-term remission from T1D by sustaining intrapancreatic Tregs and controlling Tems. A: Overtly diabetic NOD mice were randomized to receive 15 μg CD3-specific F(ab’)₂ fragment alone or in combination with oral insulin (0.5 mg twice a week for 5 consecutive weeks). The percentage of protected mice was followed in each group, and the data are presented in two separate graphs for 1) mice with a BGV between 250 and 350 mg/dL at onset or 2) the entire mouse cohort independently of BGV at onset (n = 26–42 mice per group). B: The percentage of CD44^highCD62L^low Tems within the CD4 or CD8 compartments was calculated in various organs of NOD mice either diabetic (sick) or protected for at least 15 weeks by treatment with 15 μg anti-CD3 alone (CD3) or in combination with oral insulin (CD3/insulin). Data are expressed as means ± SEM and are representative of three independent experiments with n = 4–8 mice per group. C: The percentage of CD4⁺Foxp3⁺ cells within the CD4 population was measured in various organs of protected NOD mice 15 weeks posttreatment with 15 μg anti-CD3 alone or in combination with oral insulin. Data are expressed as means ± SEM and are representative of three independent experiments with n = 5–9 mice per group. D: Total T cells were purified from the spleens or pooled PLNs and pancreata of donor NOD mice protected for at least 15 weeks by a treatment with 15 μg anti-CD3 alone or in combination with oral insulin. Equal numbers of total T cells (10⁶ cells) were transferred into NOD-SCID recipient mice, and diabetes development (BGVs >250 mg/dL) was followed. Data are representative of two independent experiments with n = 6–8 mice per group.

FIG. 5.

Synergy of anti-CD3 and oral insulin therapies augments the number of insulin-specific CD4+ T cells with in vivo suppressive capacities. Purified CD4⁺ (A) or CD4⁺CD25⁺ cells (10⁶ cells) (B) from NOD mice treated with anti-CD3 alone or anti-CD3/oral insulin (CT) and diabetogenic cells (Diab; 3 × 10⁶ cells) from fully diabetic NOD mice were cotransferred into NOD-SCID recipient mice. Autoimmune diabetes development and kinetics were followed in each group. Data are representative of two independent experiments with n = 7–8 mice per group.

FIG. 6.

Oral insulin in combination with anti-CD3 therapy increases anti-inflammatory cytokine levels expressed by insulin-specific T cells. A: Cytokine secretion by insulin-specific CD4⁺ T cells. Spleens and PLNs of protected NOD mice were collected at 4 weeks posttreatment with anti-CD3 antibody alone (CD3) or in combination with oral insulin (CD3/insulin). The number of IL-4, IL-10, and IFN-γ–expressing CD4⁺ T cells was assessed by enzyme-linked immunosorbent spot assay after in vitro stimulation with or without porcine insulin (20 μg/mL) or insB9–23 peptide (10 μg/mL). Data are expressed as means ± SEM of three independent experiments. B: CD3/oral insulin CT augments TGF-β expression by CD4⁺ T cells. Lymphocytes were recovered from various organs of NOD mice at 4 weeks posttreatment with anti-CD3 alone or in combination with oral insulin. The amount of TGF-β was measured by enzyme-linked immunosorbent assay in the supernatant of CD8-depleted cells with or without anti-CD3 stimulation. Histograms show the means ± SEM of two independent experiments. NS, nonstimulated.